Gram-positive bacteria employ surface proteins to bind to host tissues, to escape from innate and acquired immune responses, or to invade host epithelia and immune cells. Surface proteins of Staphylococcus aureus are anchored to the cell wall envelope by a mechanism requiring both an N-terminal signal peptide and a C-terminal sorting signal with an LPXTG motif. Sortase A (SrtA), a membrane anchored transpeptidase, cleaves the LPXTG motif of the sorting signal between its threonine (T) and glycine (G) residues and tethers the C-terminal carboxyl group of surface proteins to the amino group of cell wall cross-bridges within the lipid II peptidoglycan precursor. The product of this reaction is subsequently incorporated into the cell wall envelope via the transpeptidation and transglycosylation reactions of peptidoglycan synthesis. S. aureus mutants lacking the srtA gene display significant defects in the pathogenesis of animal disease. The genome of S. aureus encodes two sortase genes, each of which provide distinct anchoring mechanisms for defined sets of surface proteins. The genomes of some gram-positive microbes harbor additional sortase genes that provide for the assembly of pili on the surface of C. diphtheria, E. faecalis, S. agalactiae, A. naeslundii, C. perfringens and perhaps other pathogens. Experimental work in this research proposal tests three general hypotheses: (i) Inhibitors of sortases may be useful for the treatment of bacterial infectious diseases, (ii) The genes and gene products involved in surface protein transport and anchoring to the cell wall envelope may be identified using systematic genetic analysis, (iii) Pilus formation in gram-positive bacteria involves three unique sortase-mediated reactions that can be characterized by revealing the molecular structures of trans-peptidation reaction products.